In article <ifag.9.0 at po.uni-stuttgart.de>,
Volker Seibert <ifag at po.uni-stuttgart.de> wrote:
>Hi everyone!
>I want to express a protein from a gram (+) strain (Rhodococcus erythropolis
>1CP) in E. coli BL 21 (DE3)pLysS. I am using pET11a and pRSET6a vectors.
>Unfortunately, I get most of the protein in inclusion bodies. I have tried
>several conditions including different temperatures (25, 30 and 37øC),
>additives (betaine, sorbitol) and media (M9 and dYT). Renaturation from
>inclusion bodies was not successful. Can anyone help me?
>Thanks in advance.
>Volker
I have had similar problems in the past as well. The best solution
I found was based on work by the Burgess lab (Protein Express. and
Purif. 1:81). If you are following the standard protocol, you are
removing insolubles (including your inclusion bodies) with a high
speed spin. Proteins can be resolubilized from the inclusion bodies
by incubation for an hour on ice in a solution containing
0.25% sarkosyl, with a lot of pestling at the beginning and at
a few other times to try to break up the hard pellet. This seems
to resolubilize anywhere from around 5->50% of the protein, depending
on what protein is being used and how well you get the pellet
broken up. It doesn't denature the protein, but it does break apart
multimeric proteins (at least one that I tried). This is probably
not a problem in your case.
Dan Mytelka
mytelka at mendel.berkeley.edu